PROJECT SUMMARY Liver is a vital organ with a unique capability to regenerate after injuries via a dual mechanism. This ability, however, is significantly compromised by aging. Most studies that investigate the aging effect on liver regeneration focus on the signaling pathways that drive the cell cycle reentry and growth of regenerating hepatocytes. A missing but critical piece of the puzzle, as a recent study from my lab has shown, is the mechanism that maintains the regenerative potential of hepatocytes by safeguarding them from replication- induced DNA damage. The key driver of this program is a stem and cancer cell-enriched protein, nucleostemin (NS). To date, it remains unclear whether the NS pathway plays a determinant role in the age-associated decline of liver regeneration and how its expression and activity is regulated in the liver during the aging process. To solve the puzzle of how aging affects liver regeneration, there is a critical need to determine the mechanism by which aging regulates NS function in the liver. My long-term goal is to determine how self- renewal programs contribute to the regeneration and homeostasis of adult tissues during the aging process. The objective of this proposal is to determine the role of NS in age-associated decline of liver regeneration and how its expression is down-regulated in regenerating hepatocytes during the aging process. Our central hypothesis states that age-related NS down-regulation via a C/EBP?-controlled mechanism sets a threshold (determinant) level that limits the regenerative potential of the liver. This hypothesis is formulated based on: 1) NS is up-regulated on or before the cell cycle reentry of regenerating hepatocytes and serves an essential role in protecting them from replicative DNA damage; 2) NS deletion compromises liver regeneration after injuries; 3) the levels of NS and Ki67 are both down-regulated in aged livers under the resting and regenerating conditions compared to young livers; 4) NS promoter contains cognate C/EBP?-binding sites, and its transcriptional activity is inhibited by C/EBP?; and 5) C/EBP? is known to inhibit the regeneration of aged livers. To test our hypothesis, two specific aims will be pursued. Aim 1 will determine the role of NS in age- associated decline of liver regeneration. Aim 2 will determine the transcriptional mechanism that drives NS down-regulation during liver aging. At the completion, we expect to have determined the importance of NS in setting a threshold level that caps the regenerative potential of aged livers, and the transcriptional program that drives the down-regulation of NS during the aging process. These outcomes will fill the need to understand the molecules that underpin the age-related decline of liver regeneration and the mechanisms that may precipitate or alleviate the effect of aging on liver regeneration. A successful completion of this proposal will therefore have a direct impact on resolving the health issues caused by age-dependent decline of liver regeneration and a broad impact on advancing research on the aging effect on the regeneration of other adult tissues.